title: Glutathione depletion by valproic acid in sandwich-cultured rat hepatocytes: Role of biotransformation and temporal relationship with onset of toxicity.
abstract: The present study was conducted in sandwich-cultured rat hepatocytes to investigate the chemical basis of glutathione (GSH) depletion by valproic acid (VPA) and evaluate the role of GSH depletion in VPA toxicity. Among the synthetic metabolites of VPA investigated, 4-ene-VPA and (E)-2,4-diene-VPA decreased cellular levels of total GSH, but only (E)-2,4-diene-VPA was more effective and more potent than the parent drug. The in situ generated, cytochrome P450-dependent 4-ene-VPA did not contribute to GSH depletion by VPA, as suggested by the experiment with a cytochrome P450 inhibitor, 1-aminobenzotriazole, to decrease the formation of this metabolite. In support of a role for metabolites, alpha-F-VPA and octanoic acid, which do not undergo biotransformation to form a 2,4-diene metabolite, CoA ester, or glucuronide, did not deplete GSH. A time course experiment showed that GSH depletion did not occur prior to the increase in 2',7'-dichlorofluorescein (a marker of oxidative stress), the decrease in [2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium] (WST-1) product formation (a marker of cell viability), or the increase in lactate dehydrogenase (LDH) release (a marker of necrosis) in VPA-treated hepatocytes. In conclusion, the cytochrome P450-mediated 4-ene-VPA pathway does not play a role in the in situ depletion of GSH by VPA, and GSH depletion is not an initiating event in VPA toxicity in sandwich-cultured rat hepatocytes.
authors.affiliation: Faculty of Pharmaceutical Sciences, The University of British Columbia, Vancouver, British Columbia, Canada.
authors.forename: Tony K L
authors.initials: TK
authors.lastname: Kiang
authors.forename: Xiao Wei
authors.initials: XW
authors.lastname: Teng
authors.forename: Jayakumar
authors.initials: J
authors.lastname: Surendradoss
authors.forename: Stoyan
authors.initials: S
authors.lastname: Karagiozov
authors.forename: Frank S
authors.initials: FS
authors.lastname: Abbott
authors.forename: Thomas K H
authors.initials: TK
authors.lastname: Chang
mesh_terms: D000818:Animals; D001711:Biotransformation; D002470:Cell Survival / Q000187:drug effects; D056486:Chemical and Drug Induced Liver Injury / Q000209:etiology* / Q000378:metabolism*; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D005978:Glutathione / Q000378:metabolism*; D022781:Hepatocytes; D007770:L-Lactate Dehydrogenase / Q000032:analysis; D008297:Male; D018384:Oxidative Stress / Q000187:drug effects*; D051381:Rats; D017207:Rats, Sprague-Dawley; D013778:Tetrazolium Salts / Q000737:chemistry; D013997:Time Factors; D014635:Valproic Acid / Q000633:toxicity*
chemical_list: C110656:2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium; D013778:Tetrazolium Salts; D014635:Valproic Acid; D003577:Cytochrome P-450 Enzyme System; D007770:L-Lactate Dehydrogenase; D005978:Glutathione